Epithelial ovarian malignant neoplastic disease ( EOC ) , which compromises 90 % of all ovarian malignances, is the taking cause of gynaecological malignant neoplastic disease decease in developed states ( 1 ) . The decease rate for this disease has non changed much in the last 50 old ages.
Ovarian malignant neoplastic disease is more common in patients with elevated gonadotrophins ( FSH and LH ) , such as postmenopausal adult females or patients undergoing ovulation initiation ( 2 ; 3 ) . Additionally, reduced hazard for ovarian malignant neoplastic disease is associated with multiple gestations, breast-feeding, and unwritten preventives, which are all associated with lower degrees of and decreased exposure to gonadotrophins ( 4 ; 5 ) . The receptors for FSH and LH are present in ovarian carcinoma cells ( 6 ; 7 ) . Furthermore, the degrees of ovarian and peritoneal gonadotrophins seem to be elevated in ovarian malignant neoplastic disease patients ( 8-11 ) . Together, these observations suggest a possible function for gonadotrophins in the development and patterned advance of ovarian malignant neoplastic disease. This hypothesis is supported by recent surveies showing that intervention of ovarian malignant neoplastic disease cells with gonadotrophins in vitro consequences in an enhanced metastatic phenotype that is characterized by proteolytic activity activation and aggressive invasive behaviour ( 12 ; 13 ) . Gonadotropins besides induce the look of extra cistrons that can lend to angiogenesis or metastasis, including MMPs, vascular endothelial growing factor ( VEGF ) and Cox ( COX ) enzymes ( 12 ; 14-21 ) .
The Cox ( besides called prostaglandin endoperoxide synthase ) enzymes catalyze the stepwise transition of arachidonic acid into two ephemeral intermediates, prostaglandin G2 ( PGG2 ) and prostaglandin H2 ( PGH2 ) , which is so metabolized to assorted prostaglandins by the activity of specific synthases ( 22 ) . Two Cox isoforms have been identified and are referred to as COX-1 and COX-2. The COX-1 and COX-2 enzymes are involved in tumour patterned advance by advancing proliferation, endurance, invasion, and metastasis in several solid tumours ( 22-27 ) . In ovarian malignant neoplastic disease, elevated look of COX-1 and COX-2 has been reported late, proposing that these enzymes play a cardinal function in the patterned advance of ovarian carcinoma ( 28-33 ) .
The current survey was undertaken in order to prove the hypothesis that gonadotropins ( FSH and LH ) promote the metastatic phenotype of ovarian malignant neoplastic disease cells via a COX-dependent mechanism. Treatment of ovarian tumour cells with FSH and LH in vitro induced COX-1 and COX-2 messenger RNA and protein look in a time- and dose-dependent mode. Furthermore, gonadotrophin increased COX-1 and COX-2 look via the PI3K/ AKT signaling pathway. Inhibition of COX-1 and COX-2 decreased gonadotropin-induced PGE2 production, and MMP-2 and MMP-9 activity, and barricade gonadotropin-mediated motility and invasion. These informations show that COX-1 and COX-2 contribute to gonadotropin-mediated ovarian malignant neoplastic disease patterned advance.
Materials and Methods
Human LH and recombinant FSH were obtained from Dr. A.F. Parlow ( National Hormone and Pituitary Program, Harbor-University of California-Los Angeles Medical Center, Torrance, CA ) . COX-1 and COX-2 antibodies, SC-560, and NS-398 were purchased from Cayman Chemical ( Ann Arbor, MI ) . LY294002 was purchased from Calbiochem ( San Diego, CA ) . FSH receptor ( FSHR ) and LH receptor ( LHR ) antibodies were purchased from Santa Cruz Biotechnology ( Santa Cruz, CA ) . MMP-2 and MMP-9 monoclonal antibodies were purchased from Neomarkers ( Fremont, CA ) , and anti-AKT and anti-phospho-AKT ( Ser 473 ) were purchased from Cell Signaling Technology, Inc. ( Beverly, MA ) .
The human ovarian malignant neoplastic disease cell lines ( CaOV-3 and SKOV-3 ) were purchased from the American Type Culture Collection ( ATCC, Manassas, VA ) , and their usage was approved by the University of British Columbia Clinical Screening Committee for Research and Other Studies Involving Human Subjects. Cells were cultured in Medium 199: MCDB 105 ( 1:1 ; Sigma-Aldrich Corp. , St. Louis, MO ) incorporating 10 % foetal bovine serum ( FBS ; Hyclone Laboratories Ltd. , Logan, UT ) , 100 U/ml penicillin G and 100 g/ml streptomycin ( Life Technologies, Inc. , Rockville, MD ) in a humidified ambiance of 5 % CO2-95 % air at 37A°C. The cells were passaged with 0.06 % trypsin ( 1:250 ) / 0.01 % EDTA in Mg2+ /Ca2+ – free HBSS at meeting. For monolayer civilizations, the cell lines were maintained on tissue civilization dishes ( Falcon, Becton Dickinson, Franklin Lakes, NJ ) . Human granulosa cells ( hGC ) cells were obtained from adult females undergoing in vitro fertilisation intervention. Their usage was approved by the Research Ethics Board of the University of British Columbia. Granulosa cells from each patient were extracted as described antecedently ( 34 ) and were cultured in DMEM/F-12 ( Sigma-Aldrich Corp. ) supplemented with 10 % FBS, 100 U/ml penicillin G and 100 g/ml streptomycin in a humidified ambiance of 5 % CO2-95 % air at 37A°C.. After 48 H, the hGL cells were collected for protein extraction.
Entire RNA was prepared utilizing the TRIzol reagent ( Invitrogen Canada, Burlington, ON, Canada ) , harmonizing to the maker ‘s instructions. Single-stranded complementary DNA was synthesized from 2 Aµg of entire RNA following the maker ‘s process ( Amersham Bioscienes, Quebec, Canada ) . The undermentioned primers ( Invitrogen ) were used for PCR elaboration: COX-1, sense, 5’-TGC CCA GCT CCT GGC CCG CCG CTT-3 ‘ ; antisense, 5’-GTG CAT CAA CAC AGG CGC CTC TTC-3 ‘ ; COX-2, sense, 5’- CCC TTG GGT GTC AAA GGT AA-3 ‘ ; antisense, 5’- GCC CTC GCT TAT GAT CTG TC-3 ‘ . The comparative messenger RNA degrees were normalized utilizing glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) messenger RNA degrees, which were determined with the following specific primers: sense 5’-ATG GAA ATC CCA TCA CCA TCT T-3 ‘ ; antisense, 5’-CGC CCC ACT TGA TTT TGG-3 ‘ . The rhythm Numberss that generated half-maximal elaboration were used for subsequent semi-quantitative analysis of cistron look ; these values were 33 rhythms for COX-1 and COX-2, and 22 rhythms for GAPDH. The PCR was carried out with protocol of 30 sec at 94oC, 30 sec at 58oC and 1 min at 72oC. The PCR merchandises were analyzed by cataphoresis on a 1.5 % agarose gel and the signal strength was quantified with the National Institutes of Health ( Bethesda, MD ) ImageJ package.
Western smudge analysis
Cells were harvested in lysis buffer [ 50mM Tris ( pH 7.5 ) , 150mM NaCl, 1 % NP40, 0.5 % Na deoxycholate, 1mM EDTA, 0.1 % SDS ] that contained a peptidase inhibitor cocktail ( Sigma ) , and protein concentrations were determined utilizing the Bradford protein check ( Bio-Rad Laboratories, Inc. , Hercules, CA ) . Protein ( 40 Aµg ) was electrophoresed on a 7.5 % SDS-PAGE. To find the secernment of MMPs from ovarian malignant neoplastic disease cells, conditioned medium ( 40 Aµl ) was electrophoresed under cut downing conditions on a 7.5 % SDS-PAGE, transferred to a nitrocellulose membrane ( Amersham Pharmacia Biotech. , Oakville, ON, Canada ) , and reacted with specific primary antibodies at 4oC overnight. The signals were detected with horseradish peroxidase-conjugated secondary antibody for 1 H, and were visualized utilizing the ECL chemiluminescent system ( Amersham Pharmacia Biotech. ) and the signal strength was quantified with the ImageJ package.
Invasion and migration checks
Twenty-four-well transwell inserts with an 8-Aµm pore coated with 1mg/ml Matrigel ( 50 Aµl/well ; BD scientific disciplines, Mississauga, ON, Canada ) were used to measure cell invasion. Trypsinized cells ( 1A-105 ) in serum-free medium, with or without FSH/ LH, were seeded in triplicate in the upper chamber. 5 % FBS medium was placed in the lower Wellss. The Chamberss were incubated for 48 H at 37oC in a 5 % CO2 atmosphere. Cells that had non penetrated the filter were wiped away, and invaded cells on the lower surface of the filter were fixed with ice-cold methyl alcohol and stained with 0.5 % crystal violet. Consequences are presented as the average figure of invaded cells of five Fieldss ( at 100x magnification ) A± SEM from three independent experiments. To measure cellular migration potency, the protocol described above was used, except that Matrigel was omitted and 1 % FBS medium was placed in the lower chamber as a chemoattractant.
PGE2 enzyme-linked immunosorbent check
Degrees of PGE2 released into the medium of treated cells were analyzed by PGE2 enzyme-linked immunosorbent check ( Cayman Chemical ) harmonizing to the maker ‘s instructions. Synthesized PGE2 concentrations were normalized against protein concentrations measured in the corresponding whole-cell infusions.
To mensurate the activity of MMPs, the learned medium was incubated with non-reducing dilution buffer before cataphoresis on a 12.5 % SDS-polyacrylamide gel incorporating 0.1 % gelatin. Following cataphoresis, the gel was washed twice in 2.5 % Triton X-100 to take the SDS and was incubated overnight at 37 °C in a buffer [ 50mM Tris ( pH 7.4 ) , 0.2M NaCl, 5mM CaCl2, 0.02 % Brij 35 ] that allows both pro-gelatinase and active gelatinase to digest the gelatin. The gel was so stained with Coomassie bluish G-250 ( Bio-Rad Laboratories ) to visualise gelatinase activity. Valuess represent the comparative MMP activity ( both pro- and active-form ) and were normalized based on the measuring of feasible cell figure utilizing an MTT check.
All the experiments were performed at least three times. All values are expressed as agencies A± SEM. Data were analyzed by one-way ANOVA followed by Tukey ‘s station hoc trial utilizing GraphPad Prism 5 ( GraphPad Software, San Diego, CA ) . P & lt ; 0.05 was considered statistically important.
Consequence of gonadotrophins on COX-1 and COX-2 look in ovarian malignant neoplastic disease cells
As a first measure toward analysing the function of the COX tract in gonadotropin-mediated ovarian malignant neoplastic disease patterned advance, we investigated the consequence of gonadotrophins ( FSH and LH ) on COX-1 and COX-2 look in CaOV-3 and SKOV-3 cells, which express functional FSH and LH receptors ( Fig 1 ) . Our consequences showed that intervention with FSH and LH stimulated COX-1 and COX-2 messenger RNA look in time-dependent mode in SKOV-3 cells ( Fig 2 ) . FSH and LH intervention besides resulted in a time- and dose-dependent up-regulation of COX-1 and COX-2 protein look in CaOV-3 and SKOV-3 cells ( Fig 3A & A ; B ) .
Gonadotropins-induced COX-1 and COX-2 up-regulation via the PI3K tract in SKOV-3 cells
It is well-documented that the PI3K/ AKT tract is often amplified and serves as a survival tract in ovarian carcinomas ( 35 ; 36 ) . In add-on, gonadotrophins are known to trip the PI3K/ AKT tract in human ovarian surface epithelial cells and ovarian carcinoma cell lines ( 12 ; 37 ; 38 ) . The PI3K signaling pathway and its downstream protein, animal starch synthase kinase 3b ( GSK3I? ) , have been reported to modulate COX-1 and COX-2 look ( 39 ; 40 ) . Therefore, we analyzed whether the PI3K/ AKT tract was involved in gonadotropin-induced COX-1 and COX-2 look in SKOV-3 ovarian malignant neoplastic disease cells. We foremost examined the phosphorylation position of AKT molecules upon intervention with 100 ng/ ml FSH and LH at 5, 15, 30, 60, and 120 proceedingss post-treatment. As shown, intervention with FSH and LH stimulated the activation of AKT ( Ser 473 ) in a time-dependent mode ( Fig. 4A ) . The selective PI3K inhibitor, LY294002, markedly inhibited gonadotropin-induced COX-1 and COX-2 look ( Fig. 4C ) , demoing the significance of the PI3K/ AKT tract in gonadotropin-mediated up-regulation of COX-1 and COX-2 look in SKOV-3 cells.
Gonadotropins-induced motility and invasion are COX-1 and COX-2 dependant in SKOV-3 cells
Gonadotropins have been antecedently reported to advance the invasiveness of ovarian malignant neoplastic disease cells ( 12 ) . To find the motility of ovarian malignant neoplastic disease cells, a Transwell migration check was performed. The consequence of COX-1 and COX-2 suppression on gonadotropin-stimulated cell migration was evaluated. Our consequences showed that intervention of cells with a COX-1-specific inhibitor ( SC-560 ) or COX-2-specific inhibitor ( NS-398 ) blocked gonadotropin-stimulated migration at 24 hours ( Fig. 5A ) .
To further analyze if COX-1 and COX-2 activity is required for the invasive capacity of ovarian malignant neoplastic disease cells, a Matrigel-coated Transwells invasion check was performed. Consequences showed that the suppression of COX-1 and COX-2 activity, by SC-560 and NS-398, severally, abrogated the stimulatory consequence of gonadotrophins on CaOV-3 and SKOV-3 cell invasive activity ( Fig. 5B ) . We found that FSH and LH significantly increased the invasiveness of CaOV-3 and SKOV-3 cells and that pretreatment with LY294002, a PI3K inhibitor, markedly blocked gonadotropin-induced invasion ( informations non shown ) , which was consistent with our recent findings ( 12 ) . Taken together, COX-1 and COX-2 play indispensable functions in gonadotropin-stimulated ovarian malignant neoplastic disease cell motility.
Gonadotropin-mediated up-regulation of PGE2 production, and MMP-2 and MMP-9 are COX-1 and COX-2 dependant in SKOV-3 cells
To analyze whether the addition of COX-1 and COX-2 look is associated with elevated prostaglandin synthesis, the degree of PGE2 released from CaOV-3 and SKOV-3 cells was measured utilizing ELISA. FSH and LH increased PGE2 degrees in the learned medium of CaOV-3 and SKOV-3 cells in a COX-1- and COX-2-dependent mode ( Fig. 6A ) . These informations indicate that both enzymes participate in PGE2 production.
As antecedently reported, FSH and LH stimulate the production of MMP-2 and MMP-9 ( 12 ) . Following, we asked whether COX-1 and COX-2 mediate the gonadotropin-induced activity of MMP-2 and MMP-9. SKOV-3 cells were pretreated with COX-1 ( SC-560 ) and COX-2 ( NS-398 ) specific inhibitors and were so treated with FSH/ LH for an extra 48 hours. MMP-2 and MMP-9 activities were evaluated utilizing gelatin zymography. Treatment with SC-560 and NS-398 blocked the ability of FSH and LH to bring on both pro-form and active-form of MMP-2 and MMP-9, implicating COX-1 and COX-2 as go-betweens of gonadotropin-mediated MMP-2 and MMP-9 activation ( Fig. 6B ) . This consequence was confirmed by Western blotting, which showed that gonadotropin-mediated MMP-2 and MMP-9 secernment was inhibited by SC-560 and NS-398 ( Fig. 6C ) . These consequences suggest that COX-1 and COX-2 are required for gonadotropin-enhanced PGE2 production, and MMP-2 and MMP-9 secernment and activity.
PGE2 stimulates cell invasion, and MMP-2 and -9 activities in SKOV-3 cells
Several lines of grounds indicate that COX-derived PGE2 besides plays an of import function in the invasive belongingss of human malignant neoplastic disease cells ( 41-45 ) . Therefore, we examined the consequence of PGE2 on cell invasion and MMP activity in ovarian malignant neoplastic disease cells. CaOV-3 and SKOV-3 cells were treated with increasing concentrations of PGE2, resulted in a dose-dependent biphasic ordinance on invasion: cells treated with PGE2 at low doses of 0.1nM to 10nM showed significantly higher Numberss of invaded cells, whereas the response to higher doses of PGE2 ( 100nM ) was undistinguished ( Fig. 7A ) . We besides examined whether the consequence of PGE2 on ovarian malignant neoplastic disease invasion was associated with altered MMP activity and production. As shown in Fig. 7B and 7C, intervention with the PGE2 induced a biphasic ordinance form for MMP activity and secernment in SKOV-3 cells. These informations indicate that COX-derived PGE2 may lend to gonadotropin-induced cell invasiveness in ovarian malignant neoplastic disease.
The presence of gonadotrophins in the ovarian tumour fluid of ovarian malignant neoplastic disease patients suggests the importance of these factors in the transmutation and patterned advance of ovarian malignant neoplastic diseases ( 8-11 ) . The observation of high FSH and LH degrees in the cyst fluid of malignant ovarian tumours is consistent with the gonadotrophin theory. Because the hypothesis that pituitary gonadotrophins increase the hazard of ovarian malignance and that gestations and unwritten preventives protect the ovary by stamp downing secernment of these endocrines was antecedently suggested ( 46 ) , legion surveies have examined the function of gonadotrophins on ovarian malignant neoplastic disease. Gonadotropins promote the proliferation, endurance, and metastasis of ovarian malignant neoplastic disease by bring oning the look of cardinal regulative cistrons ( 47 ) . Proteinase look is besides modulated by gonadotrophins in ovarian malignant neoplastic disease cells, taking to gonadotropin-dependent alterations in motility and invasive behaviour ( 12 ) . The present survey shows that gonadotrophins besides induce COX-1 and COX-2 look, and, in analogue, PGE2 production in ovarian carcinoma cells and, for the first clip, implicates COX-1 and COX-2 as downstream go-betweens of gonadotropin-induced effects on tumour patterned advance in human ovarian malignant neoplastic disease cells.
Data sing COX-2 look in the normal ovary suggest that it is a constituent of the ovulatory procedure ( 48-50 ) while repeated ovulations were suggested to do malignant transmutation of ovarian epithelial tissue. Recent epidemiological and clinical surveies suggest that the COX-1 and COX-2 enzymes play a cardinal function in the patterned advance of ovarian carcinoma ( 28-33 ) . Treatment with FSH and LH/ human chorionic gonadotrophin ( human chorionic gonadotropin ) has been shown to up-regulate the look of COX-1 and COX-2 in different human cells and tissues ( 14-17 ; 19 ; 51 ) . In the current survey, intervention of ovarian tumour cells with FSH and LH in vitro induced COX-1 and COX-2 messenger RNA and protein looks in a time- and dose-dependent mode. These informations indicate that FSH and LH may advance ovarian carcinoma patterned advance through their consequence on the look of the COX-1 and COX-2 enzymes.
In ovarian malignant neoplastic disease, the PI3K/ AKT signaling pathway plays a function in proliferation, antiapoptosis, migration, invasion, and metastasis ( 52 ; 53 ) . Emerging grounds suggests the engagement of the PI3K/ AKT signaling pathway in ordinance of COX-1 and COX-2 look ( 40 ; 54 ; 55 ) . Gonadotropins may do the activation of PI3K and AKT in other generative tissues, including farinaceous cells, Sertoli cells, and oocytes ( 56-58 ) . As for ovarian malignant neoplastic diseases, we showed antecedently that both FSH and LH non merely increased EGFR degrees in human OSE cells ( 37 ) , but besides activated proteolysis and increased invasion in ovarian malignant neoplastic disease cells through the PI3K/ AKT signaling tract ( 12 ) . The current informations support a function for gonadotrophins in the initiation of COX-1 and COX-2 look, at least in portion, via the PI3K/ AKT tract in ovarian carcinoma cells. The activation of the PI3K/ AKT tract was confirmed by the increased phosphorylation of AKT in the presence of FSH and LH ( Fig. 4A ) . In understanding with our old survey ( 12 ) , FSH and LH-induced activation of AKT was wholly abolished by pretreatment with LY294002, a PI3K inhibitor ( Fig 4B ) . Aberrant suppression of the PI3K signaling pathway besides blocked gonadotropin-induced stimulation of COX-1 and COX-2 look in ovarian carcinoma cells ( Fig. 4C ) .
The ability of COX-1-specific and COX-2-specific inhibitors to cut down gonadotropin-mediated motility and invasion indicates that COX-1 and COX-2 play a major function in ovarian tumour cell motility and invasion. There is increasing grounds that COX-1 and COX-2 contribute to tumor patterned advance by advancing tumour cell proliferation, angiogenesis and invasion ( 23 ; 32 ; 45 ; 54 ) . Furthermore, overexpression of COX-2 elevated tumorigenicity, tumour growing and invasion of human epidermoid carcinoma cells via up-regulated MMP ( 59 ) . Similarly, COX-2-overexpressing colon carcinoma cells exhibit enhanced pro-MMP-2 activation and invasiveness that is blocked by intervention with a COX inhibitor ( 60 ) while COX-2 suppression leads to decreased MMP-2 look in different malignant neoplastic disease cells, including ovarian malignant neoplastic disease cells ( 61-63 ) . We have reported antecedently that both FSH and LH enhanced ovarian malignant neoplastic disease cell invasiveness via the activation and/or look of MMP-2 and MMP-9 ( 12 ) . Our current information show that the add-on of specific COX-1 and COX-2 inhibitors blocked gonadotropin-induced MMP-2 and MMP-9 look and activity, showing the cardinal function of COX-1 and COX-2 in the gonadotropin-induced MMP-dependent motility and invasiveness of ovarian carcinoma cells.
The ability of COX-specific inhibitors to cut down gonadotropin-induced PGE2 production indicates that both COX-1 and COX-2 drama of import functions in PGE2 formation in ovarian malignant neoplastic disease cells. There is increased grounds that PGE2 besides contributes to tumor patterned advance by advancing cell invasion and that this consequence is mediated, at least in portion, by the transition of MMP activity ( 45 ) . We reported antecedently ( 12 ) that gonadotropin stimulates ovarian malignant neoplastic disease cell invasion and MMP activity. Here, we demonstrated that, in SKOV-3 cells, gonadotropin-induced PGE2 production is involved in cell invasion and MMP activity, bespeaking that gonadotropin-induced cell invasion may be regulated via COX-dependent PGE2 signaling.
In decision, the present consequences implicate gonadotrophins in the initiation of COX-1 and COX-2 look, which so mediate downstream effects on ovarian malignant neoplastic disease patterned advance. Given that these two enzymes contribute to PGE2 production in these tumours, the possibility of suppressing COX- and PGE2-related signaling Cascadess may hold relevant deductions in the bar and intervention of this malignance. Take together, our consequences indicate that gonadotrophins may increase COX-dependent invasion by triping the PI3K tract. Because ovarian malignant neoplastic disease patterned advance involves metastasis and is more common in conditions with high degrees of gonadotrophins, understanding the cellular and molecular mechanisms behind the function of gonadotrophins in invasion and/or metastasis may assist clarify the etiology of ovarian malignant neoplastic disease development.
This work was supported by the Canadian Institutes of Health Research ( P C K L ) and the Hong Kong Research Grants Council ( A S T W ) . P C K L is receiver of a Distinguished Scientist Award and M T L is receiver of Graduate Studentship Awards from the Interdisciplinary Women ‘s Reproductive Health Research Training Program.
Figure 1. Expression of FSHR and LHR in ovarian malignant neoplastic disease cells. Western smudge analysis of FSHR, LHR and i??-actin degrees in CaOV-3, SKOV-3 cells and the positive control cells, human granulose cells ( hGC ) .
Figure 2. Consequence of gonadotrophins on COX-1 and COX-2 messenger RNA look in SKOV-3 cells. ( A ) SKOV-3 cells were treated with 100 ng/ml of FSH/ LH for 0 to 24 H as indicated. COX-1 and COX-2 messenger RNA initiation was analyzed by semiquantitative PCR. Primers for elaboration of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) messenger RNA were used as controls. Results represent the mean A± SEM ( n=3 ; * , P & lt ; 0.05 ; ** , P & lt ; 0.001 ) .
Figure 3. Consequence of gonadotrophins on COX-1 and COX-2 protein look in ovarian malignant neoplastic disease cells. ( A ) SKOV-3 cells were treated with 100 ng/ml of FSH/ LH for 0 to 24 H as indicated and analyzed by Western blotting utilizing COX-1 and COX-2 antibodies. ( B ) CaOV-3 and SKOV-3 cells were cultured for 24 H in the presence or absence of FSH/ LH as indicated. COX-1 and COX-2 protein degrees were analyzed by Western blotting. I?-actin antibody was used as a control for equal burden. Results stand for the mean A± SEM ( n=3 ; * , P & lt ; 0.05 ; ** , P & lt ; 0.001 ) .
Figure 4. Gonadotropins bring on COX-1 and COX-2 look via the PI3K/AKT signaling pathway. ( A ) SKOV-3 cells were treated with 100 ng/ml of FSH/ LH for 0 to 120 min as indicated and AKT look was analyzed by Western blotting utilizing phospho-AKT ( Ser 473 ) and AKT antibodies. ( B & A ; C ) SKOV-3 cells were pretreated with 10 AµM of LY294002 for 30 min before add-on of FSH/ LH ( 100 ng/ml ) for 30min ( B ) or 24 H ( C ) . COX-1, COX-2, phosphor-AKT and AKT protein degrees were analyzed by Western blotting. I?-actin antibody was used as a control for equal burden. Results stand for the mean A± SEM ( n=3 ; * , P & lt ; 0.05 ; ** , P & lt ; 0.001 ) .
Figure 5. COX-1 and COX-2 inhibitors suppress gonadotropin-induced migration and invasion. ( A ) Ovarian malignant neoplastic disease cells were seed in uncoated filter ( 8i?m pore ) of Transwell Chamberss and preincubated for 30min with 50 nanometers of SC-560 or 10 AµM of NS-398 before intervention with 100 ng/ml of FSH/ LH for 24 H, as indicated. ( B ) Cell invasion under same intervention for 48h was measured through Matrigel-precoated filters. Results represent the mean A± SEM ( n=3 ; ** , P & lt ; 0.001 ) .
Figure 6. COX-1 and COX-2 inhibitors suppress gonadotropin-induced PGE2 production, and MMP-2 and MMP-9 look and activity. SKOV-3 cells were pretreated for 30 min with 50 nanometers of SC-560 or 10 AµM of NS-398 before intervention with 100 ng/ml of FSH/ LH for 24 H ( A ) or 48 H ( B and C ) , as indicated. ( A ) PGE2 production from SKOV-3 cells was analyzed by PGE2 ELISA. PGE2 concentrations normalized to matching cell protein content. a, P & lt ; 0.05, as compared with the control ( C ) ; B, P & lt ; 0.05, as compared with FSH ; degree Celsiuss, P & lt ; 0.05, as compared with LH. ( B ) Enzymatic activity of MMP-2 and MMP-9 was studied in learned medium by SDS-PAGE gelatin zymography as described in Materials and Methods. Arrows indicate gelatinase activity matching to pro-MMP-2, active MMP-2, pro-MMP-9, and active MMP-9. Results represent the mean A± SEM ( n=3 ; * , P & lt ; 0.05 ; ** , P & lt ; 0.001 ) . The values were normalized based on the measuring of feasible cell figure utilizing an MTT check. ( C ) Conditioned medium was analyzed by Western blotting utilizing MMP-2 and MMP-9 antibodies. Results represent the mean A± SEM ( n=3 ; * , P & lt ; 0.05 ) . The values were normalized based on the measuring of feasible cell figure utilizing an MTT check.
Figure 7. PGE2 induced SKOV-3 cell invasion, and MMP-2 and MMP-9 look and activity. SKOV-3 cells were treated with ( 0.01nM- 100nM ) PGE2 for 24 H, as indicated. ( A ) SKOV-3 cells were seeded in Matrigel-coated invasion Chamberss in the presence of PGE2 for 24 H, as indicated. Enzymatic activity ( B ) and look ( C ) of MMP-2 and MMP-9 from SKOV-3 cells were studied as described in Fig. 5. Result represent the average A± SEM ( n=3 ; * , P & lt ; 0.05 ; ** , P & lt ; 0.001 ) .